Cell lines have played an important role in the development of molecular and cellular biology, particularly in the elucidation of intracellular activities, the effects of extracellular molecules and cell-cell interactions. Cell lines are established stepwise by: explantation of tissue containing a heterogeneous cell population; separation of the cells; isolation of a cell clone; and culturing the cell clone so that the total cell number increases over several generations and the population is uniform in its lineage. For instance, cell cultures may be started from primary tissue culture explants, where heterogeneous cell types separate or migrate from the tissue in liquid medium; or by enzyme digestion of a tissue, resulting in dispersed cell suspensions.
Differentiation is the process of maturation of cells. It is a progressive and dynamic process, beginning with pluripotent stem cells and ending with terminally differentiated cells that progress no further down the cell lineage pathway. A cell's function, phenotype and growth characteristics are affected by the cell's degree of differentiation.
Cells that can be continuously cultured are known as immortalized cells. Immortalized cells have advantages over non-immortalized cells because they can be cultured to provide large numbers of uniform cell populations. Immortalized cells are routinely used for understanding intracellular activities, such as the replication and transcription of DNA, metabolic processes and drug metabolism. Investigation of cellular transmembrane activities, such as, ligand-receptor interactions and signal transduction, is facilitated by access to specific cell types. Immortalized cells are also useful in examining specific cell-cell interactions, such as adhesion, invasion and contact inhibition. However, many cell types have remained recalcitrant to isolation and continuous culture. In addition, many differentiated cells lose some of their differentiated properties (dedifferentiate) in order to regain or retain the ability to proliferate. Thus, if an immortalized cell line were available that could be continuously cultured it would not necessarily express the differentiation functions that make it a valuable tool.
One such cell type that has been difficult to immortalize is the dendritic cell, an antigen presenting cell, and its precursors that are at early stages of differentiation. Steinman et al., WO 93/20185, have disclosed methods for isolating primary dendritic cells and their precursors from tissue. Granucci et al., WO 94/28113, and Paglia et al., J. Exp. Med. 178:1893-1901, 1993, have disclosed cell lines isolated from primary cultures and infected with retroviral vectors to immortalize the cells.
Dendritic cells are the most potent antigen presenting cells (APCs) in the immune system. Dendritic cells are the only cells that present antigen to, and activate, naive CD4.sup.+ T cells in vivo (Levin et al., J. Immunol. 151:6742-6750, 1993). Dendritic cells are found in primary and secondary lymphoid organs (e.g., thymus, lymph nodes, tonsils, Peyer's patches, and spleen), as well as in non-lymphoid organs and tissues (e.g., heart, liver, lung, gut, and in the skin as epidermal Langerhans cells). Dendritic cells are also prevalent in afferent lymph, but are rare in blood. For reviews, see Steinman, Ann. Rev. Immunol. 9:271-296, 1991 and Knight et al., J. Invest. Dermatol. 99:33S-38S, 1992.
Dendritic cells are thought to originate from a single hematopoietic progenitor cell. As progenitor cells begin the process of differentiation they migrate to selected tissue and/or organs, where they appear to undergo additional differentiation. If isolated from tissue, dendritic cells are immature; that is, the cells are not fully differentiated, are inefficient at antigen presentation, express low levels of MHC Class II molecules and do not stimulate proliferation of T-cells in an allogenic mixed leukocyte reaction (MLR). However, when immature dendritic cells are exposed to foreign proteins, they become capable of taking up and presenting soluble antigen via newly synthesized MHC Class II molecules, and stimulataneously leave their tissue residence and migrate to lymph nodes and spleen. After migrating from the origin tissue, the dendritic cells are mature; that is, they exhibit high levels of MHC Class II, accessory and co-stimulatory molecules, as well as full APC function (Steinman, ibid., 1990 and Ibrahim et al., Immunol. Today 16:181-186, 1995). Antigen uptake and processing by dendritic cells are not well understood, however, because of the inability to isolate and culture sufficient numbers of homogeneous dendritic cells or dendritic precursor cells.
Dendritic cells have been implicated as the primary causative cells in a number of different diseases that involve immune responses, including contact sensitivity, tumor immunity, HIV-1 infection and autoimmunity (e.g., Type I diabetes, multiple sclerosis and rheumatoid arthritis). These cells are believed to play a role in graft rejection, where cells from the allograft migrate into the lymphoid organs of the recipient and initiate a deleterious immune response.
Therefore, a need remains in the art for immortalized dendritic cells that can be directly isolated from a tissue source and cultured continuously. There also remains a need for immortalized dendritic precursor cells that can be activated into antigen presenting cells that retain their differentiated properties while continuously being cultured.